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Tetracycline-resistant (Tet r ) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet r gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet r genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet r strains transferred Tet r genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet r strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.Many different kinds of antibiotics have been used as therapeutic agents in aquaculture in Japan. Intensive work was done until the 1980s to develop guidelines for antibiotic usage in fish farms. The guidelines regulated doses and required a period of drug-free rearing before sale of fish and succeeded in keeping the residual antibiotics in cultured fish to nondetectable levels. However, Samuelsen et al. (35) found that antibiotic-resistant bacteria persisted in fish farm sediments for at least 18 months after chemotherapy. Since the products of aquaculture are consumed by humans and since many antibiotic resistance determinants are encoded by transferable plasmids, cultured fish may serve as a vehicle for transmission of antibiotic resistance to bacteria that are commensal or pathogenic to humans (34).Tetracyclines are among the therapeutic agents most commonly used in human and veterinary treatment. Oxytetracycline is permitted to be mixed with feed for fish, and food sanitation law in Japan permits certain residual levels in fish. Because of the widespread use of tetracycline, resistance to it has been disseminated to many species of marine bacteria (4,6,18,42,46).More than 30 different kinds of tetracycline resistance determinants have been published. Resistance genes have been mainly categorized into two major groups, those responsible for proton-dependent efflux of tetracycline (24) and those conferring ribosomal protection by cytoplasmic proteins (9). Dissemination of the proton-dependent tetracycline efflux protein in aquaculture environments has been reported (5,12,13,14,22,34,37). Previous work has identified the relevant genes by using DNA hybridization or PCR methods (4,12,13,14,15,16,27,28,34,37), but the nucleotide sequences of these determinants remain unkn...

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Sixteen strains of aerobic bacteria which contain bacteriochlorophyll a were isolated from the samples collected in aerobic marine environments: thalli of Enteromorpha linza, Porphyra sp., Sargussum horneri; beach sand; and the surface seawater from Aburatsubo Inlet. When they occurred, their proportions among the aerobic heterotrophic populations ranged from 0.9 to 1.1% in the seaweed samples and from 1.2 to 6.3% in the beach sand samples and were 0.9% in the seawater sample. The results suggested that the aerobic photopigmented bacteria widely inhabit aerobic marine environments.

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Six strains of multidrug-resistant Stenotrophomonas maltophilia were isolated from cultured yellowtail. The strains were divided into two clusters based on the 16S rRNA genes, and all of them contained L1 metallo-␤-lactamase and L2 ␤-lactamase genes. Differences in the intercluster divergence between the lactamase genes suggest that horizontal transfer of the genes occurred.Stenotrophomonas maltophilia is a multidrug-resistant bacterium that has been isolated from numerous terrestrial environments (3,7,12,18) and recently has emerged as a nosocomial pathogen in clinical environments (8,13,19,30). Although the phylogenetic relationship between environmental and clinical strains has been examined, no clear distinction has been established yet.We isolated six strains of S. maltophilia, which were identified with API 20NE (BioMerieux, La Balm, France), from yellowtail (Seriola quinqueradiata) cultured in a fish farm located at the southern tip of Japan (14). All six strains grew on Muller-Hinton agar (Becton Dickinson Microbiology Systems, Cockeysville, MD) supplemented with either ampicillin (32 g ml Ϫ1 ) or panipenem (32 g ml Ϫ1 ). The strains were also resistant to cefotaxime and ceftazidime, as determined by susceptibility tests with Sensi-Disks (Showa, Tokyo, Japan). Although the strains grew in a seawater-based medium, the growth rates in this medium were 0.6-fold lower than those with no NaCl. Hence, we concluded that the strains are not indigenous to marine environments. The resistance to ampicillin seemed to be a factor that could induce the presence of S. maltophilia in the fish farm, since medication with ampicillin was used in the previous year according to the fish farmer. Samuelsen et al. have reported persistence of drug-resistant bacteria even in the absence direct selection (28). NaCl may have been a factor in the survival. Resistance to panipenem, cefotaxime, and ceftazidime probably did not play a role in the distribution of the strains, since none of these antibiotics can be used in aquaculture. Rather, the resistance to these drugs suggests that the strains were derived from clinical environments.16S rRNA gene sequences of the six strains were determined for the phylogenetic analysis by a method described previously (14). The phylogenetic analysis divided the strains into two distinct clusters, cluster A (strains BL-9, BL-10, BL-15, and BL-16) and cluster B (strains BL-12 and BL-13), and both of the clusters were included in group I described by Hauben et al., which is comprised mainly of clinical and environmental strains (15).PCR amplification with forward primer 5Ј-CACACCTGGC AGATCGGCAC-3Ј and reverse primer 5Ј-GCCGCATCCGC GTAGGC-3Ј at an annealing temperature of 65°C was used to amplify L1 metallo-␤-lactamase (L1), which can degrade carbapenems (23,24,33). L2 serine ␤-lactamase (L2), which cannot degrade carbapenems (23, 32), was PCR amplified with forward primer 5Ј-CGATTCCTGCAGTTCAGT-3Ј and reverse primer 5Ј-CGGTTACCTCATCCGATC-3Ј at an annealing temperature of 55°C. No differences were foun...

A rapid clustering scheme for Vibrio specieswas developed based on the analysis of 16S rDNA, which compriseda group‐specific polymerase chain reaction (PCR) and restriction fragmentlength polymorphisms (RFLP) of the PCR‐amplified 16S rDNA. The group‐specificPCR, using a specific primer Vib1F, clustered the Vibrio speciesinto two large groups: Vib1F+ group and Vib1F– group.The PCR‐RFLP, using ScaI and BlnI, further dividedthese groups into smaller groups. Finally, 46 Vibrio speciesand eight related species could be clustered into 14 groups usingthis rapid clustering method. Seasonal dynamics of the Vibrio communityin Yoshimi Bay, Hibiki‐nada Sea, Japan, were examined based on therapid clustering method. In both seawater and sediments, the groupcomprised Vibrio parahaemolyticus, Vibrio alginolyticus, Vibriocampbellii, Vibrio carchariae, Vibrio harveyi and Vibrionatriegens and was predominant when the seawater temperaturewas above 20°C, whereas the group of Vibrio splendidus biotypeI and Vibrio lentus was abundant when the temperature wasaround or below 20°C.